This invention relates to a microfabricated fluidic device, and in particular, to such a device that is configured for forming and transporting a series of minute volume segments of a material and for storage, retrieval, and analysis thereof and to a method for forming, transporting, storing, and retrieving such a series of minute volume segments.
A number of elementary microfabricated fluidic devices have been demonstrated over the past few years. Although many of these fluidic devices are quite simple, they are demonstratively powerful in addressing many realistic applications and may well revolutionize the way that biochemical separations are performed. The majority of the demonstrations have involved transferring known chemical measurement techniques, such as electrophoresis or liquid chromatography, onto microfabricated platforms. Such demonstrations suggest that microfabricated separation devices will be quite useful for improving the time and cost associated with collecting information from such experiments. However, the known devices have not exploited the new experimental approaches that such microfabricated devices potentially enable. We believe that through improvements in microfluidic control, new more powerful biochemical experimental paradigms will arise.
The area of microfabricated fluidics that has received the most attention is electrokinetically driven processes. Electrokinetic fluid manipulations have been demonstrated for mixing and reacting reagents, injection or dispensing of samples, and chemical separations. Electrically driven separation techniques such as capillary electrophoresis (CE), open channel electrochromatography (OCEC) and micellar electrokinetic capillary chromatography (MEKC) have been demonstrated by a number of research groups. Both dsDNA fragments and sequencing products have been sized using microchip capillary gel electrophoresis coupled with laser induced fluorescence detection. Less conventional electrophoretic separations have been studied in post arrays using DC and pulsed electric fields. In addition fluorescence-based competitive immunoassays have been demonstrated using microchip electrophoretic separation of bound and free labeled antigen. These miniature devices have shown performance either equivalent to or better than conventional laboratory devices in all cases investigated and appear to offer the rare combination of xe2x80x9cbetter-faster-cheaperxe2x80x9d simultaneously. Microchip separation devices exhibit speed advantages of one to a few orders of magnitude over conventional approaches. The efficiency of electrophoretic separations under diffusion limited conditions is proportional to the voltage drop experienced by the sample. These diffusion limiting conditions can be achieved for short separation distances on microchips due to the narrow axial extent of the injection plugs that are generated. The time of analysis decreases quadratically with separation distance at constant applied potential, which gives a fundamental advantage to microchip- based electrophoretic separations.
Other significant advantages of microchip based chemical separations are the small volumes that can be analyzed, the ability to monolithically integrate sample processing and analysis, and the low cost of replication which makes possible highly parallel analyses. All of these factors are consistent with high throughput analysis and reductions in cost and time to generate biochemical information. Early efforts demonstrating integration of sample processing include post-separation and pre-separation derivatization of amino acids coupled to electrophoretic separations. On-chip DNA restriction digestions and PCR amplifications have been coupled with electrophoretic fragment sizing on integrated monolithic microchips. Cell lysis, multiplex PCR, and CE analysis were performed on plasmic-containing E. coli cells in a single device. Parallel PCR/CE assays of multiple samples in chips containing multiple reaction wells have also been demonstrated. In addition, competitive immunoassay experiments have been performed on a microchip device that included fluidic elements for mixing of sample with reagents, incubation, and electrophoretic separations. Other microfabricated fluidic elements that have been coupled to electrically driven separations include electrospray ionization for analysis by mass spectrometry, and sample concentration using porous membrane elements and solid phase extraction. Devices have also been demonstrated that employ electrokinetic transport solely for performing chemical and biochemical reactions. Examples include devices for enzymatic reaction kinetics, enzyme assays, organic synthesis, and cellular manipulations. All four of these latter applications could eventually be of significant importance to experimental biology, but have not been sufficiently developed at this time.
A number of microfabricated fluidic devices have also been demonstrated that use hydraulic forces for fluid transport. While the use of hydraulic forces can be applied to a broader range of fluidic materials than electrokinetic phenomena, it is less convenient to implement in general. External connections to microchips for hydraulically driven flow are more cumbersome than applying an electric potential. Moreover, electrokinetically driven forces follow the flow of electrical current and thus, allow greater control over transport within a microchannel manifold versus application of pressure or vacuum to a terminus of such a manifold. Electrokinetic forces are also able to generate much higher effective pressures than is practical with hydraulics. The demonstrated capabilities of hydraulically driven devices appear to be trailing that of electrokinetically driven devices. Nonetheless, a number of important capabilities have been reported.
Microfluidic devices for performing PCR have received considerable interest. Early devices included only chambers machined in silicon to act as sample reservoirs while later devices utilized the silicon structure for resistive heating or utilized integrated filters for the isolation of white blood cells. More recently, an interesting device for continuous flow PCR was reported that utilized a single microchannel that meanders through temperature zones to accomplish thermal cycling. Filters for processing cellular materials have been micromachined into silicon substrates. Flow cytometry devices have also been micromachined into silicon and glass substrates and driven hydraulically.
The capability to manipulate reagents and reaction products xe2x80x9con-chipxe2x80x9d suggests the eventual ability to perform virtually any type of xe2x80x9cwet-chemicalxe2x80x9d bench procedure on a microfabricated device. The paradigm shift of moving the laboratory to a microchip includes the advantages of reducing reagent volumes, automation or material manipulation with no moving parts, reduced capital costs, greater parallel processing, and higher processing speed. The volume of fluids that are manipulated or dispensed in the microfluidic structures discussed above is on the nanoliter scale or smaller versus tens of microliters at the laboratory scale, corresponding to a reduction of three orders of magnitude or more. Flow rates on the devices that have been studied are of the order of 1 mL/yr of continuous operation. By implementing multiple processes in a single device (vertical integration), these small fluid quantities can be manipulated from process to process efficiently and automatically under computer control. An operator would only have to load the sample to be analyzed. Obviously, this serial integration of multiple analysis steps can be combined with parallel expansion of processing capacity by replicating microfabricated structures, e.g., parallel separation channels, on the same device.
Although the so-called xe2x80x9cLab-on-a-Chipxe2x80x9d appears to hold many promises, and it is believed that it will fulfill many of them, there are further developments necessary to achieve an impact level that parallels the scale of miniaturization realized in the field of microelectronics. There are at least four significant issues that must be addressed to bring xe2x80x9cLab-on-a-Chipxe2x80x9d devices to the next level of sophistication, or processing power, over the next decade. Those issues are: advanced microfluidic control, the xe2x80x9cworld-to-chipxe2x80x9d interface, detection, and viable manufacturing strategies. Presently, electrokinetic manipulation of fluids in microchannel structures represents the state-of-the-art in controlled small volume handling with high precision. The strategy has been to control the time-dependent electric potential at each of the channel terminals to move materials along a desired path. While this strategy has been very effective for valving and mixing in simple designs, it is limited in its applicability and performance as designs become more complex. We believe that new strategies that allow control of electric potentials at multiple points in the microchannel design will be necessary for these more complex structures. Electrokinetic transport also has limitations in the types of solutions and materials that can be manipulated.
The world-to-chip interface is the term we have assigned to the problem of delivering multiple samples or reagents onto microchips to achieve high throughput analysis. Although a given sample can be analyzed in times as brief as a millisecond, multiple samples cannot presently be presented to microchip devices at such a rate. There has been very little effort directed toward this problem, but it represents a major hurdle to achieving ultrahigh throughput experimentation.
In accordance with a first aspect of the present invention, there is provided a method of forming and transporting a series of minute volume segments (nanoliter or subnanoliter) of a material on a fluidic microchip, wherein each of the volume segments are separated by segmenting material. The method includes the steps of providing a first channel having an inlet end connected to a source of transport fluid and an outlet end connected to a fluid reservoir and providing a second channel having an inlet end connected to a source of segmenting fluid and an outlet end interconnected with said first channel. A minute volume of the segmenting fluid is drawn into the first channel and transported in the first channel toward the fluid reservoir. The steps of drawing and transporting the minute volume of segmenting fluid into the first channel are repeated to form a series of transport fluid volumes and/or analysis volumes between the segmenting fluid volumes. Reagents, cells, or reaction beads can be injected or inserted into the transport fluid volumes to provide a series of assay or analysis volumes. The assay or analysis volumes are transported in series fashion so as to provide serial registration thereof for storage and retrieval for later analysis or other manipulation on the microchip.
In accordance with another aspect of this invention, there is provided an apparatus for forming and transporting a series of minute volume segments of a material. The device is a fluidic microchip having first and second microchannels formed on a substrate. The first microchannel has an inlet end connected to a source of transport fluid and an outlet end connected to a fluid reservoir. The second microchannel has an inlet end connected to a source of segmenting fluid and an outlet end interconnected with the first microchannel. A plurality of electrodes are disposed in the first microchannel between the outlet ends of the first microchannel and the second microchannel. A second plurality of electrodes are disposed in said first microchannel between the inlet end of said first microchannel and said second microchannel. The device further includes means for (i) inserting a volume of the segmenting fluid from the second microchannel into the first microchannel, thereby displacing the transport fluid contained in the first microchannel, (ii) stopping the transport of the segmenting fluid volume in the first microchannel, and then (iii) transporting the interleaved transport and segmenting volumes in the first microchannel toward the fluid reservoir.
Further embodiments of the device according to this invention include additional channels, sources of reagents, reagent diluents, cells, and/or reaction particles, for inserting such materials into transport volumes formed by sequential pairs of the segmenting fluid volumes. Means for transporting the reagents, cells, and/or reaction beads are also included in such embodiments. In a preferred arrangement, the first microchannel includes one or more loops to provide serial storage of the reaction volumes for later retrieval and analysis or manipulation.